Manuel Hervas - Academia.edu (original) (raw)

Papers by Manuel Hervas

Physiologia Plantarum

Cytochrome c is an extrinsic component in the luminal side of photosystem II in cyanobacteria, as... more Cytochrome c is an extrinsic component in the luminal side of photosystem II in cyanobacteria, as well as in eukaryotic algae from the red photosynthetic lineage including, among others, diatoms. We have established that cytochrome c from the diatom Phaeodactylum tricornutum can be obtained as a complete protein from the membrane fraction of the alga, although a C-terminal truncated form is purified from the soluble fractions of this diatom as well as from other eukaryotic algae. Eukaryotic cytochromes c show distinctive electrostatic features as compared with cyanobacterial cytochrome c . In addition, co-immunoseparation and mass spectrometry experiments, as well as immunoelectron microscopy analyses, indicate that although cytochrome c from P. tricornutum is mainly located in the thylakoid domain of the chloroplast -where it interacts with photosystem II-, it can also be found in the chloroplast pyrenoid, related with proteins linked to the CO concentrating mechanism and assimilation. These results thus suggest new alternative functions of this heme protein in eukaryotes. This article is protected by copyright. All rights reserved.

Photosynthesis research, Jan 28, 2016

The photosynthetic cytochrome c 550 from the marine diatom Phaeodactylum tricornutum has been pur... more The photosynthetic cytochrome c 550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c 550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c 550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c 550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c 550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c 550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c 550 decreases up to about 45% as compared to iron...

Plant and Cell Physiology, 2016

Cyt, cytochrome; Cox, cytochrome c oxidase; E m , midpoint redox potential; Em,7, midpoint redox ... more Cyt, cytochrome; Cox, cytochrome c oxidase; E m , midpoint redox potential; Em,7, midpoint redox potential at pH 7; GFP, green fluorescent protein; k bim , second-order rate constant; k inf , second-order rate constant extrapolated to infinite ionic strength; k obs , observed pseudo-first-order rate constant; ORF, open reading frame; Pc, plastocyanin; pI, isoelectric point; PSI, photosystem I.

Frontiers in Plant Science, 2016

Photosynthesis Research, Feb 1, 2002

The role played by the residues Leu12 and Lys33 - which are both located at the north hydrophobic... more The role played by the residues Leu12 and Lys33 - which are both located at the north hydrophobic patch of plastocyanin - in the interaction of the copper protein with Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 has been investigated by site-directed mutagenesis. A thermodynamic analysis of PS I reduction by wild-type and mutant plastocyanins has been performed by laser-flash absorption spectroscopy. In all cases, the electron transfer is impaired by mutations, which induce drastic changes in the apparent activation entropy of the overall reaction. Substitution of Leu12 by alanine specifically affects the hydrophobic interactions with PS I, whereas replacement of Lys33 by glutamate not only induces local electrostatic changes, but also alters the hydrophobic interactions with the photosystem. The thermodynamic analysis of the reactivity of K33E mutant towards PS I reveals that the effect of the mutation can be reversed by addition of magnesium cations, which probably bind at a place close to Glu33. The electrostatic surface potential does thus modulate the hydrophobic interactions with PS I by altering the solvent accessibility of some surface residues.

Photosynthesis Research, 2001

Cytochrome c 6 (Cyt) from the thermophilic cyanobacterium Phormidium laminosum has been purified ... more Cytochrome c 6 (Cyt) from the thermophilic cyanobacterium Phormidium laminosum has been purified and characterized. It is a mildly acidic protein, with physicochemical properties very similar to those of plastocyanin (Pc). This is in agreement with the functional interchangeability of the two metalloproteins as electron donors to Photosystem I (PS I). The kinetic analyses of the interaction of Pc and Cyt with Photosystem I show that both metalloproteins reduce PS I with similar efficiencies, according to an oriented collisional kinetic model involving repulsive electrostatic interactions. The thermostability study of the Phormidium Pc/PS I system compared with those from mesophilic cyanobacteria (Synechocystis, Anabaena and Pseudanabaena) reveals that Pc is the partner limiting the thermostability of the Phormidium couple. The cross-reactions between Pc and PS I from different organisms demonstrate not only that Phormidium Pc enhances the stability of the Pc/PS I system using PS I from mesophilic cyanobacteria, but also that Phormidium PS I possesses a higher thermostability than the other photosystems. Abbreviations: Cyt-Cytochrome c 6 ; k bim-bimolecular rate constant for the overall reaction; k inf-bimolecular rate constant extrapolated to infinite ionic strength; Pc-plastocyanin; PS I-Photosystem I; P700-the photoactive dimeric chlorophyll molecule in Photosystem I; T i-temperature of inflection of the experimental kinetic data for PS I reduction compared with the Eyring plot; T m-midpoint temperature of protein thermal transition; T maxtemperature at which the maximum rate for PS I reduction is observed; G ‡ , H ‡ and S ‡-changes in apparent activation free energy, enthalpy and entropy, respectively, for the overall reaction of PS I reduction

Photosynthesis Research, 2003

European Journal of Biochemistry, 1990

In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochrome c552 ... more In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochrome c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavin, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate that cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20-100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome ~5 5 2 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavincytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed.

Journal of Inorganic Biochemistry, 1995

Journal of Biological Chemistry, 2001

Positively charged plastocyanin from Anabaena sp. PCC 7119 was investigated by site-directed muta... more Positively charged plastocyanin from Anabaena sp. PCC 7119 was investigated by site-directed mutagenesis. The reactivity of its mutants toward photosystem I was analyzed by laser flash spectroscopy. Replacement of arginine at position 88, which is adjacent to the copper ligand His-87, by glutamine and, in particular, by glutamate makes plastocyanin reduce its availability for transferring electrons to photosystem I. Such a residue in the copper protein thus appears to be isofunctional with Arg-64 (which is close to the heme group) in cytochrome c 6 from Anabaena

Journal of Biological Chemistry, 2012

Background: Plant NTRC contains a flavin, a disulfide group, and an extra thioredoxin module. Res... more Background: Plant NTRC contains a flavin, a disulfide group, and an extra thioredoxin module. Results: The flavin cofactor in NTRC is oxidized following two intramolecular reactions that are altered by peroxiredoxin. Conclusion: In comparison with canonical NTRs, NTRC shows additional conformational dynamics affected by peroxiredoxin. Significance: NTRC is involved in the response to oxidative stress and in maintaining the redox homeostasis of plastids. NADPH-dependent thioredoxin reductases (NTRs) contain a flavin cofactor and a disulfide as redox-active groups. The catalytic mechanism of standard NTR involves a large conformational change between two configurations. Oxygenic photosynthetic organisms possess a plastid-localized NTR, called NTRC, with a thioredoxin module fused at the C terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs) and thus is involved in the protection against oxidative stress, among other functions. Although the mechanism of electron transfer of canonical NTRs is well established, it is not yet known in NTRC. By employing stopped-flow spectroscopy, we have carried out a comparative kinetic study of the electron transfer reactions involving NTRC, the truncated NTR module of NTRC, and NTRB, a canonical plant NTR. Whereas the three NTRs maintain the conformational change associated with the reductive cycle of catalysis, NTRC intramolecular electron transfer to the thioredoxin module presents two kinetic components (k ET of ϳ2 and 0.1 s ؊1), indicating the occurrence of additional dynamic motions. Moreover, the dynamic features associated with the electron transfer to the thioredoxin module are altered in the presence of 2-Cys Prx. NTRC shows structural constraints that may locate the thioredoxin module in positions with different efficiencies for electron transfer, the presence of 2-Cys Prx shifting the conformational equilibrium of the thioredoxin module to a specific position, which is not the most efficient. * This work was supported by Andalusian Government European Regional Development Fund-cofinanced Grants BIO-022 and CVI-4528 (to J. A. N.) and European Regional Development Fund-cofinanced Grant BIO2010-15430 from Ministry of Science and Innovation and Andalusian Government Grant BIO-182 and CVI-5919 (to F. J. C.

Eukaryotic Cell, 2005

Recently, two types of fatty acid synthases (FASs) have been discovered from apicomplexan parasit... more Recently, two types of fatty acid synthases (FASs) have been discovered from apicomplexan parasites. Although significant progress has been made in characterizing these apicomplexan FASs, virtually nothing was previously known about the activation and regulation of these enzymes. In this study, we report the discovery and characterization of two distinct types of phosphopantetheinyl transferase (PPTase) that are responsible for synthesizing holo-acyl carrier protein (ACP) from three apicomplexan parasites: surfactin production element (SFP) type inCryptosporidium parvum(CpSFP-PPT), holo-ACP synthase (ACPS)-type inPlasmodium falciparum(PfACPS-PPT), and both SFP and ACPS types inToxoplasma gondii(TgSFP-PPT and TgACPS-PPT). CpSFP-PPT and TgSFP-PPT are monofunctional, cytosolic, and phylogenetically related to animal PPTases. However, PfACPS-PPT and TgACPS-PPT are bifunctional (fused with a metal-dependent hydrolase), likely targeted to the apicoplast, and more closely related to proteo...

Biochemistry, 1995

The reaction mechanism of electron transfer from the interchangeable metalloproteins plastocyanin... more The reaction mechanism of electron transfer from the interchangeable metalloproteins plastocyanin (Pc) and cytochrome c6 (Cyt) to photooxidized P700 in photosystem I (PSI) has been studied by laser-flash absorption spectroscopy using a number of evolutionarily differentiated organisms such as cyanobacteria (Anabaena sp. PCC 7119 and Synechocystis sp. PCC 6803), green algae (Monoraphidium braunii), and higher plants (spinach). PSI reduction by Pc or Cyt shows different kinetics depending on the organism from which the photosystem and metalloproteins are isolated. According to the experimental data herein reported, three different kinetic models are proposed by assuming either an oriented collisional reaction mechanism (type I), a minimal two-step mechanism involving complex formation followed by intracomplex electron transfer (type II), or rearrangement of the reaction partners within the complex before electron transfer takes place (type III). Our findings suggest that PSI was able to first optimize its interaction with positively charged Cyt and that the evolutionary replacement of the ancestral Cyt by Pc, as well as the appearance of the fast kinetic phase in the Pc/PSI system of higher plants, would involve structural modifications in both the donor protein and PSI.

Biochemical and Biophysical Research Communications, 1998

The genes coding for plastocyanin (petE) and cytokaryotic algal species that are able to form eit... more The genes coding for plastocyanin (petE) and cytokaryotic algal species that are able to form either Cyt chrome c 6 (petJ) from Anabaena sp. PCC 7119 have or Pc depending on copper availability in the culture been cloned and properly expressed in Escherichia medium (3). The two proteins are acidic in eukaryotes coli. The recombinant proteins are identical to those and either neutral or basic in cyanobacteria, but they purified from the cyanobacterial cells. The products of both exhibit a similar isoelectric point when isolated both the petE and petJ genes are correctly processed from the same organism (3, 4). In filamentous cyanoin E. coli, as deduced from their identical N-terminal bacteria such as Anabaena, Pc and Cyt are positively amino acid sequences as compared with those of the charged with an isoelectric point of ca. 9 (see below). metalloproteins isolated from the cyanobacterium. Whereas a great deal of structural information exists Physicochemical and functional properties of the nafor Pc from eukaryotic organisms (see ref. 5, for a retive and recombinant protein preparations are also view), 3D structures of cyanobacterial Pc have been identical, thereby confirming that expression of petE reported only very recently: first, the solution structure and petJ genes in E. coli is an adequate tool to address of Pc from Anabaena variabilis as solved by NMR specthe study of the structure/function relationships in troscopy (6); and second, the X-ray structure of a triple plastocyanin and cytochrome c 6 from Anabaena by site-directed mutagenesis. ᭧ 1998 Academic Press mutant Pc from Synechocystis sp. PCC 6803 (7). Re-Key Words: Anabaena; cytochrome c 6 : petE gene; petJ gards Cyt, its crystal and solution structure has only gene; plastocyanin. been reported in eukaryotic algae (8-11). From all these structural data, it is clear that Pc and Cyt posses primary and secondary structures that are completely different, but they exhibit a number of common surface Plastocyanin (Pc) 3 and cytochrome c 6 (Cyt) are two features that account for their functional interchangesmall redox proteins that function as mobile electron ability (2). carriers between the two membrane-embedded com-Recent laser flash kinetic analyses indicate that the plexes cytochrome b 6 f and Photosystem I (PSI) in oxyredox interaction between the two metalloproteins and genic photosynthesis (see refs. 1 and 2, for recent re-PSI follows three different reaction mechanisms of inviews). Some less-evolved cyanobacteria synthesize creasing complexity (4). In Anabaena, in particular, just Cyt and higher plants produce only Pc, but there PSI reduction kinetics can be well fitted to a simple oriented collisional mechanism with Pc but to a more complex and efficient three-step model with Cyt, the 1 EMBL Accession Numbers AJ002361 for the pet J gene and latter involving complex formation, rearrangement of AJ002362 for the pet E gene.

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1994

Laser flash absorption spectroscopy has been used to investigate the kinetics of electron transfe... more Laser flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from reduced cytochrome c6 and plastocyanin to photooxidized P700 in Photosystem I (PS I) particles from the cyanobacterium Synechocystis PCC 6803. Data analysis yields second-order rate constants of 1.3.107 M-1 s-1 and 1.0.107 M-1 s-1 for the heme-and copper-proteins, respectively. With the two donor proteins, the observed rate constants (kob s) present a linear protein-concentration dependence, thus suggesting an apparent one-step bimolecular collisional mechanism. At neutral pH, the kob s values monotonically increase with increasing NaC1 or MgC12 concentration, which is ascribed to the involvement of repulsive electrostatic interactions between the donor proteins and PS I. The difference in the effective concentration at which MgCI 2 has its maximum effect as compared with that of NaC1 is attributed to the specific role played by Mg 2+ ions, which could act as electrostatic bridges between negatively charged groups. At physiological mild acid pH, cytochrome c 6 is a more efficient electron donor than plastocyanin. The inversion of the NaC1 and MgC12 effect at pH below 5-that is, decreasing of kob s with increasing ionic strength-is interpreted as arising from the involvement of attractive ionic interactions at pH lower than the isoelectric point of the donor proteins. Some evolutive aspects on the mechanism of electron donation to PS I are discussed.

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1989

Selective light-induced redox changes of cytochrome b-559 in relation to cytochrome f have been i... more Selective light-induced redox changes of cytochrome b-559 in relation to cytochrome f have been investigated at 20 °C in fresh spinach thylakoids under physiological conditions and in the presence of specific inhihitors and uncouplers. Light absorbed by PS II (650 nm) promotes the sequential reduction of cytochrome b-559 and cytochrome f, whereas light absorbed by PS I (720 nm) brings about the oxidation of cytochrome f and, by way of this, the oxidation of cytochrome b-559. Either DCMU or DBMIB completely blocks the reduction of the two cytochromes by PS II light, but not the oxidation of cytochrome f by PS I light. On the other hand, cyanide as well as polylysine inhibits almost totally the oxidation of both cytochromes by PS I light. From these and previous results, it is concluded that photoreduction of cytochrome b-559 by PS II occurs through the plastoquinone pool and that its photooxidation by PS 1 involves both plastocyanin and cytochrome f. It thus appears that cytochrome b-559 is located between plastoquinone and cytochrome f in noncyclic electron transport in chloroplasts.

Archives of Biochemistry and Biophysics, 1995

$Research paper thumbnail of Cytochrome c 6 from the green alga Monoraphidium braunii . Crystallization and preminary diffraction studies$

Acta Crystallographica Section D Biological Crystallography, 1995

Cytochrome c(6), a plastocyanin functionally interchangeable electron carrier between the chlorop... more Cytochrome c(6), a plastocyanin functionally interchangeable electron carrier between the chlorophyll molecule P700 of photosystem I and cytochrome f from cytochrome b(6)f complex, has been isolated from the green alga Monoraphidium braunii and crystallized by the vapour-diffusion technique in sodium citrate. Crystals belong to space group R3, with cell dimensions a = b = 51.93 (5) and c = 80.5 (1) A (hexagonal axes), with one molecule per asymmetric unit. They diffract beyond 1.9 A under a Cu Kalpha rotating-anode source, with an anomalous signal that allows the positioning of the heme Fe atom in the unit cell.